Nutritional intervention for improving muscular function and strength

ABSTRACT

The present invention provides a composition comprising HMB and Vitamin D. Methods of administering HMB and Vitamin D to an animal are also described. Vitamin D and HMB are administered to increase muscle mass, strength, and functionality. The combination of Vitamin D and HMB together has a synergistic effect, which results in a surprising and unexpected level of improvement in muscle mass, strength and functionality.

This application is a continuation of U.S. patent application Ser. No.14/869,533, filed Sep. 29, 2015, which is a continuation-in-part of U.S.patent application Ser. No. 14/861,728, filed Sep. 22, 2015, which is acontinuation-in-part of U.S. patent application Ser. No. 14/219,522,filed Mar. 19, 2014, which is a continuation-in-part of U.S. patentapplication Ser. No. 12/634,507, filed Dec. 9, 2009, which claims thebenefit of U.S. Provisional Application No. 61/121,065, filed Dec. 9,2008, and herein incorporates U.S. Provisional Application No.61/121,065 by reference.

BACKGROUND OF THE INVENTION

1. Field

The present invention relates to a composition comprisingβ-hydroxy-β-methylbutyrate (HMB) and Vitamin D, and methods of using acombination of HMB and Vitamin D to improve muscle mass, strength, orfunctionality.

2. Background

HMB

The only product of leucine metabolism is ketoisocaproate (KIC). A minorproduct of KIC metabolism is β-hydroxy-β-methylbutyrate (HMB). HMB hasbeen found to be useful within the context of a variety of applications.Specifically, in U.S. Pat. No. 5,360,613 (Nissen), HMB is described asuseful for reducing blood levels of total cholesterol and low-densitylipoprotein cholesterol. In U.S. Pat. No. 5,348,979 (Nissen et al.), HMBis described as useful for promoting nitrogen retention in humans. U.S.Pat. No. 5,028,440 (Nissen) discusses the usefulness of HMB to increaselean tissue development in animals. Also, in U.S. Pat. No. 4,992,470(Nissen), HMB is described as effective in enhancing the immune responseof mammals. U.S. Pat. No. 6,031,000 (Nissen et al.) describes use of HMBand at least one amino acid to treat disease-associated wasting.

It has previous been observed that HMB alone or in combination withother amino acids is an effective supplement for restoring musclestrength and function in young athletes. Further, it has been observedthat HMB in combination with two amino acids, glutamine and lysine, iseffective in increasing muscle mass in elderly persons.

HMB is an active metabolite of the amino acid leucine. The use of HMB tosuppress proteolysis originates from the observations that leucine hasprotein-sparing characteristics (1-4). The essential amino acid leucinecan either be used for protein synthesis or transaminated to theα-ketoacid (α-ketoisocaproate, KIC)(1, 3). In one pathway, KIC can beoxidized to HMB. Approximately 5% of leucine oxidation proceeds via thesecond pathway (5). HMB is superior to leucine in enhancing muscle massand strength. The optimal effects of HMB can be achieved at 3.0 gramsper day, or 0.38 g/kg of body weight per day, while those of leucinerequire over 30.0 grams per day (3).

Once produced or ingested, HMB appears to have two fates. The first fateis simple excretion in urine. After HMB is fed, urine concentrationsincrease, resulting in an approximate 20-50% loss of HMB to urine (4,6). Another fate relates to the activation of HMB to HMB-CoA (7-16).Once converted to HMB-CoA, further metabolism may occur, eitherdehydration of HMB-CoA to MC-CoA, or a direct conversion of HMB-CoA toHMG-CoA (17), which provides substrates for intracellular cholesterolsynthesis. Several studies have shown that HMB is incorporated into thecholesterol synthetic pathway (12, 16, 18-20) and could be a source fornew cell membranes that are used for the regeneration of damaged cellmembranes (3). Human studies have shown that muscle damage followingintense exercise, measured by elevated plasma CPK (creatinephosphokinase), is reduced with HMB supplementation within the first 48hrs. The protective effect of HMB lasts up to three weeks with continueddaily use (21-23).

In vitro studies in isolated rat muscle show that HMB is a potentinhibitor of muscle proteolysis (24) especially during periods ofstress. These findings have been confirmed in humans; for example, HMBinhibits muscle proteolysis in subjects engaging in resistance training(4). The results have been duplicated in many studies (25) (21-23,26-28).

The molecular mechanisms by which HMB decreases protein breakdown andincreases protein synthesis have recently been reported (29-31, 31-33).In mice bearing the MAC16 cachexia-inducing tumor, HMB attenuatedprotein degradation through the down-regulation of key activators of theubiquitin-proteasome pathway (30). Furthermore, HMB attenuatedproteolysis-inducing factor (PIF) activation and increased geneexpression of the ubiquitin-proteasome pathway in murine myotubes,thereby reducing protein degradation (31). PIF inhibits proteinsynthesis in murine myotubes by 50% and HMB attenuates this depressionin protein synthesis (29). Eley et al demonstrated that HMB increasesprotein synthesis by a number of mechanisms, including thedown-regulation of eukaryotic initiation factor 2 (eIF2) phosphorylationthrough an effect on dsRNA-dependant protein kinase (PKR) andupregulation of the mammalian target of rapamycin/70-kDa ribosomal S6kinase (mTOR/p70^(S6k)) pathway. The net result is increasedphosphorylation of 4E-binding protein (4E-BP1) and an increase in theactive eIF4G·eIF4E complex. Leucine shares many of these mechanisms withHMB, but HMB appears to be more potent in stimulating protein synthesis(29).

HMB can also increase protein synthesis by attenuating the commonpathway that mediates the effects of other catabolic factors such aslipopolysaccharide (LPS), tumor necrosis factor-α/interferon-γ(TNF-α/IFN-γ), and angiotensin II (Ang II) (32, 33). HMB acts byattenuating the activation of caspases-3 and -8, and the subsequentattenuation of the activation of PKR and reactive oxygen species (ROS)formation via down-regulation of p38 mitogen activated protein kinase(p38MAPK). Increased ROS formation is known to induce proteindegradation through the ubiquitin-proteasome pathway. HMB accomplishesthis attenuation through the autophosphorylation PKR and the subsequentphosphorylation of eIF2α, and in part, through the activation of themTOR pathway.

Numerous studies have shown an effective dose of HMB to be 3.0 grams perday as CaHMB (˜38 mg/kg body weight-day⁻¹). This dosage increases musclemass and strength gains associated with resistance training, whileminimizing muscle damage associated with strenuous exercise (34) (4, 23,26). HMB has been tested for safety, showing no side effects in healthyyoung or old adults (35-37). HMB in combination with L-arginine andL-glutamine has also been shown to be safe when supplemented to AIDS andcancer patients (38).

Studies in humans have also shown that dietary supplementation with 3grams of CaHMB per day plus amino acids attenuates the loss of musclemass in various conditions such as cancer and AIDS. (3, 4, 26, 34, 39,40) A meta-analysis of supplements to increase lean mass and strengthwith weight training showed HMB to be one of only 2 dietary supplementsthat increase lean mass and strength with exercise (34). More recentlyit was shown that HMB and the amino acids arginine and lysine increasedlean mass in a non-exercising, elderly population over a year-longstudy.

Vitamin D

Vitamin D has classically been associated with calcium and phosphorousmetabolism and bone strength. Until recently, an adequate Vitamin Dlevel has been defined using the Vitamin D deficiency disease rickets.While 1,25OH₂-VitD₃ is the active metabolite of Vitamin D, a measure ofVitamin D status widely accepted is serum (blood) circulating25OH-VitD3. A circulating blood level between 10 and 15 ng 25OH-VitD3/mLwill cause rickets in young children and has been accepted as thedeficiency level for Vitamin D. Vitamin D can be synthesized by humanswith adequate sun exposure or can be obtained through the diet andthrough supplements to the diet. Many factors influence the amount andeffectiveness of Vitamin D found in the body. These factors includedietary intake, sun exposure, Vitamin D receptor number (VDR),conversion rate from cholecalciferol to 25OH-VitD3 and finally theconversion of 25OH-VitD3 to 1,25OH₂-VitD₃.

Most of the population in northern latitudes (most of the United States)do not produce Vitamin D in the winter regardless of sun exposurebecause the sun's ultraviolet B rays do not reach the earth during thattime and therefore the only source of Vitamin D is dietary (42). As the25 hydroxylation occurs in the liver and the 1 hydroxylation occursprimarily in the kidney, these two organs play a large role indetermining the circulating levels of Vitamin D, and the functioning ofthese organs and thus Vitamin D status tends to decrease with age (42).

In a recent review, Holick details research showing that circulatinglevels of 25OH-VitD3 must reach as high as 30-40 ng/mL beforeparathyroid hormone (PTH) levels begin to plateau (43). Otherresearchers have found that increasing 25OH-VitD3 from 20 to 32 ng/mLincreased intestinal calcium transport (44). Both of these criteriawould point to a 25OH-VitD3 level of 30 ng/mL or greater being requiredfor optimal regulation of calcium metabolism in the body. A recentreview by Heaney describes the optimal level of 25OH-VitD3 to be 32ng/mL or greater for optimal health which takes into account a number ofaspects other than bone health and calcium metabolism (45). By thesestandards, from 40 to 100% of independent elderly men and women areVitamin D deficient (43).

The 1-alpha, 25-Vitamin D hydroxylase in the kidney has been consideredthe primary source for synthesis of the circulating active metabolite ofVitamin D, 1,25OH₂-VitD₃. The activity of this enzyme is regulated on awhole body level by parathyroid hormone (PTH). Regulating 1,25OH₂-VitD₃on a whole body level probably does not provide for optimal levels ofthe active vitamin for all body tissues at one time. Relatively recentlytissue specific 1-alpha, 25-Vitamin D hydroxylases have been identifiedand are thought to mediate autocrine responses of Vitamin D at thetissue specific level (46, 47). Human vascular smooth muscle has1-alpha, 25-Vitamin hydroxylase activity with a Km of 25 ng/mL. Thismeans that the enzyme is operating at one half maximal capacity at a25OH-VitD3 concentration of 25 ng/mL (48). Therefore serum levels of >25ng/mL may be necessary for optimal active Vitamin D for vascular smoothmuscle cells.

Muscle strength declines with age and a recently characterizeddeficiency symptom of Vitamin D is skeletal muscle weakness (43).Deficiency of Vitamin D and its hormonal effect on muscle mass andstrength (sarcopenia) has been described as a risk factor in falls andbone fractures in the elderly (49). Loss of muscle strength has beencorrelated with a loss of Vitamin D receptors (VDR) in muscle cells(50). Supplemental Vitamin D of at least 800 IU per day may result in aclinically significant increase in VDR in muscle cells which may be inpart be the mechanism whereby other studies have shown improvement inbody-sway, muscle strength and falling risk were seen with Vitamin Dsupplementation at this level (51). While this muscular weaknessassociated with Vitamin D may not be surprising at classical Vitamin Ddeficiency levels (blood 25OH-VitD₃ of <15 ng/mL), Bischoff-Ferrari etal continued to see improvement in lower extremity function up to andbeyond 40 ng 25OH-VitD3/mL which are levels well above what previouslymight have been thought necessary for maximal benefit (52). Thisobservation has been confirmed by other researchers that in fact minimalVitamin D levels necessary to prevent rickets do not allow for maximalphysical performance (53). A recent editorial in American Journal ofClinical Nutrition stated that all the literature available wouldindicate a 25OH-VitD₃ level of at least 30 ng/mL is most optimal forhealth and disease (54).

While the exact mechanism is still unclear, it is clear that both theactive metabolite, 1,25OH₂-VitD₃ and its precursor, 25OH-VitD3, play asignificant role in normal functioning of muscle. Muscle contains VDRsfor 1,25OH₂-VitD₃, found in both the nucleus and at the cell membrane(55-57) and these are also involved in non-specific binding 25OH-VitD3as well (58). Studies by Haddad and Birge, published in the 1970s, showthat feeding D₃ to vitamin D deficient rats 7 hours prior to measurementincreased protein synthesis as measured by ³H-leucine incorporation intomuscle cell proteins. However, when the muscles were removed from theVitamin D deficient rats and studied, only 25-OH Vit D₃ acts directly inthe muscles (58-60).

Early clinical evidence pointed to a reversible myopathy associated withVitamin D deficiency (61). Vitamin D receptors were discovered in muscletissue, thus providing direct evidence of Vitamin D's effect on musclefunction (51, 62). Muscle biopsies in adults with Vitamin D deficiencyexhibit mainly type II muscle fiber atrophy (63). Type II fibers areimportant because they are the first initiated to prevent a fall. Arecent randomized controlled study found that daily supplementation of1,000 IU of Vitamin D₂ in elderly stroke survivors resulted in anincrease in mean type II fiber diameter and in percentage of type IIfibers (64). There was also a correlation between serum 25OH-VitD3 leveland type II fiber diameter.

Vitamin D conveys its action by binding to VDR. VDR is expressed inparticular stages of differentiation from myoblast to myotubes (55, 65,66). Two different VDRs have been described. One is located at thenucleus and acts as a nuclear receptor and the other is located at thecell membrane and acts as a cellular receptor. VDR knockout mice arecharacterized by a reduction in both body weight and size as well asimpaired motor coordination (67). The nuclear VDR is a ligand-dependentnuclear transcription factor that belongs to the steroid-thyroid hormonereceptor gene superfamily (68). Bischoff et al (69) reported the firstin situ detection of VDR in human muscle tissue with significantassociated intranuclear staining for VDR. Once 1,25OH₂-VitD₃ binds toits nuclear receptor, it causes changes in mRNA transcription andsubsequent protein synthesis (70). The genomic pathway has been known toinfluence muscle calcium uptake, phosphate transport across the cellmembrane, phospholipid metabolism, and muscle cell proliferation anddifferentiation. 1,25OH-VitD₃ regulates muscle calcium uptake bymodulating the activity of calcium pumps in sacroplasmic reticulum andsacrolemma (61). Modifications of calcium levels impact muscle function(71). In vitro experiments support these findings by demonstrating anincreased uptake of ⁴⁵Ca in cells exposed to physiological levels of1,25OH₂-VitD₃ (72). The calcium binding protein calbindin D-9K issynthesized as a result of activation of nuclear VDR (62). 1,25OH₂-VitD₃plays a role in regulating phosphate metabolism in myoblasts byaccelerating phosphate uptake and accumulation in cells. 1,25OH₂-VitD₃acts rapidly, presumably through cell membrane VDRs (56, 57). Whilebinding to these receptors, there is an activation of second-messengerpathways (G-proteins, cAMP, inositol triphosphate, arachidonic acid)(73-75) or the phosphorylation of intracellular proteins. These would inturn activate protein kinase C (PKC), leading to release of calcium intomuscle cells, and ultimately resulting in active transport of Ca intothe sacroplasmic reticulum by Ca-ATPase. This process is important formuscle contraction. Additionally, PKC affects enhancements of proteinsynthesis in muscle cells (76). Recent data (77) indicate that1,25OH-VitD3 has a fast activation of mitogen-activated protein kinase(MAPK) signaling pathways, which in turn forward signals to theirintracellular targets that effect the initiation of myogenesis, cellproliferation, differentiation, or apoptosis.

Vitamin D may also regulate formation and regeneration of tightjunctions and neuromuscular junctions. In vitro studies that found thatVitamin D regulates expression of VDR and the neural growth factor (NGF)in Schwann cells (78). Recent studies have shown that Vitamin D enhancesglial cell line-derived neurotrophic factor (GDNF) in rats and that thismay have beneficial effects in neurodegenerative disease (79).Therefore, Vitamin D could act through several mechanisms of cellularfunction and neural interaction to improve overall muscle strength andfunction.

A need exists for a composition and methods to increase muscle mass andimprove function and strength. The present invention comprises acomposition and methods of using a combination of Vitamin D and HMB thatresults in such an increase in muscle mass and improvement in strengthand function.

SUMMARY OF THE INVENTION

One object of the present invention is to provide a composition forincreasing muscle mass, strength, or functionality.

Another object of the present invention is to provide methods ofadministering a composition for increasing muscle mass, strength orfunctionality.

These and other objects of the present invention will become apparent tothose skilled in the art upon reference to the following specification,drawings, and claims.

The present invention intends to overcome the difficulties encounteredheretofore. To that end, a composition comprising HMB and Vitamin D isprovided. The composition is administered to a subject in need thereofto increase muscle mass, strength and functionality. All methodscomprise administering to the animal HMB and Vitamin D.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1a is a graph showing the changes in muscle mass in subjectsdepending on Vitamin D status.

FIG. 1b is a graph showing overall knee strength based on Vitamin Dstatus with administration of HMB.

FIG. 2a is a graph showing serum Vitamin D levels in subjects.

FIG. 2b is a graph showing knee extension strength in subjects.

FIG. 2c is a graph showing an eight week performance index in subjects.

FIG. 3 shows the Protein:DNA ratio and protein degradation in C₂C₁₂cells.

FIG. 4a is a graph showing serum Vitamin D levels in subjects.

FIG. 4b is a graph showing urine HMB excretion in subjects.

DETAILED DESCRIPTION OF THE INVENTION

The present invention comprises a combination of HMB and Vitamin D thathas a synergistic effect and improves overall muscle strength andfunction. The combination of HMB and Vitamin D results in significantenhancements in overall muscle mass, function and strength. Thiscombination can be used on all age groups seeking enhancement in overallmuscle mass, function and strength. The following methods describe andshow increased overall muscle mass, function and strength even innon-exercising animals.

One specific use HMB and Vitamin D is in the older or elderlypopulation. Current estimates place a large portion of the olderpopulation at risk for falls with potential significant associatedmorbidities. The combination of HMB and Vitamin D specifically targetsmuscle mass, strength and function and consequently may producesignificant improvement in health, quality of life, and in particular,decreased falls and injury in this group.

The younger population also benefits from the administration of HMB andVitamin D, in part due to the widespread occurrence of Vitamin Ddeficiency. Women also benefit from the administration of HMB andVitamin D as women are prone to Vitamin D deficiency.

Newborn babies and children twelve months and younger can benefit fromthe administration of HMB and Vitamin D. Baby formula is Vitamin Dfortified, and the American Academy of Pediatrics (AAP) recommends thatall infants, children and adolescents take in enough Vitamin D throughsupplements, formula or cow's milk to prevent complications fromdeficiency of this vitamin.

The present invention provides a composition comprising HMB and VitaminD. The composition is administered to an animal in need of improvementin overall muscle mass, strength or function.

The composition of HMB and Vitamin D is administered to an animal in anysuitable manner. Acceptable forms include, but are not limited to,solids, such as tablets or capsules, and liquids, such as enteral orintravenous solutions. Also, the composition can be administeredutilizing any pharmaceutically acceptable carrier. Pharmaceuticallyacceptable carriers are well known in the art and examples of suchcarriers include various starches and saline solutions. In the preferredembodiment, the composition is administered in an edible form. Thecomposition of HMB and Vitamin D includes administration of thecomposition as baby formula and nutrition drinks for all ages.

B-hydroxy-β-methylbutyric acid, or β-hydroxy-isovaleric acid, can berepresented in its free acid form as (CH₃)₂(OH)CCH₂COOH. The term “HMB”refers to the compound having the foregoing chemical formula, in bothits free acid and salt forms, and derivatives thereof. While any form ofHMB can be used within the context of the present invention, preferablyHMB is selected from the group comprising a free acid, a salt, an ester,and a lactone. HMB esters include methyl and ethyl esters. HMB lactonesinclude isovalaryl lactone. HMB salts include sodium salt, potassiumsalt, chromium salt, calcium salt, magnesium salt, alkali metal salts,and earth metal salts.

Methods for producing HMB and its derivatives are well-known in the art.For example, HMB can be synthesized by oxidation of diacetone alcohol.One suitable procedure is described by Coffman et al., J. Am. Chem. Soc.80: 2882-2887 (1958). As described therein, HMB is synthesized by analkaline sodium hypochlorite oxidation of diacetone alcohol. The productis recovered in free acid form, which can be converted to a salt. Forexample, HMB can be prepared as its calcium salt by a procedure similarto that of Coffman et al. in which the free acid of HMB is neutralizedwith calcium hydroxide and recovered by crystallization from an aqueousethanol solution. The calcium salt of HMB is commercially available fromMetabolic Technologies, Ames, Iowa.

CaHMB has historically been the preferred delivery form of HMB.Previously, numerous obstacles existed to both extensive testing andcommercial utilization of the free acid form of HMB, and since it wasthought there was no difference between the two forms from apharmacokinetic perspective, the calcium salt was adopted as acommercial source of HMB. Until recently packaging and, in particular,distribution of dietary supplements has been better suited to handlenutrients in a powdered form and therefore the calcium salt of HMB waswidely accepted. HMB-acid is a liquid and much more difficult to deliveror incorporate into products.

Currently, the manufacturing process for HMB has allowed for HMB freeacid to be produced in a purity that allows for oral ingestion of theHMB free acid. Besides having a commercial source that is pure enoughfor oral ingestion, the HMB-acid needs to be buffered for oralingestion, a process which only recently was determined due to thefactors listed above which precluded previous use of HMB-acid.

It was assumed that ingestion of CaHMB would result in a rather quickdissociation of HMB from the calcium salt form. However, a recent studyand corresponding patent application (U.S. App. Publication No.20120053240) has shown that HMB in the free acid form has rather uniquepharmacokinetic effects when compared to CaHMB ingestion. Use of HMBfree acid (also called HMB-acid) improves HMB availability to tissuesand thus provides a more rapid and efficient method to get HMB to thetissues than administration of CaHMB.

Vitamin D is present in the composition in any form. In the preferredembodiment, Vitamin D₃ (cholecalciferol) is administered, but theinvention is not limited to that form of Vitamin D. While Vitamin D₃ isthe synthesized and preferred form of Vitamin D for mammals, mammals canalso use supplemental Vitamin D₂. Vitamin D2 may be less potent thanVitamin D₃, hence additional D₂ may be required in order to raise bloodlevels of 25-OH VitD₂.

When the composition is administered orally in an edible form, thecomposition is preferably in the form of a foodstuff or pharmaceuticalmedium, more preferably in the form of a foodstuff. Any suitablefoodstuff comprising the composition can be utilized within the contextof the present invention. In order to prepare the composition as afoodstuff, the composition will normally be blended with the appropriatefoodstuff in such a way that the composition is substantially uniformlydistributed in the foodstuff. Alternatively, the composition can bedissolved in a liquid, such as water. Although any suitablepharmaceutical medium comprising the composition can be utilized withinthe context of the present invention, preferably, the composition isblended with a suitable pharmaceutical carrier, such as dextrose orsucrose, and is subsequently tabulated or encapsulated as describedabove.

Furthermore, the composition can be intravenously administered in anysuitable manner. For administration via intravenous infusion, thecomposition is preferably in a water-soluble non-toxic form. Intravenousadministration is particularly suitable for hospitalized patients thatare undergoing intravenous (IV) therapy. For example, the compositioncan be dissolved in an IV solution (e.g., a saline or glucose solution)being administered to the patient. Also, the composition can be added tonutritional IV solutions, which may include amino acids and/or lipids.The amounts of the composition to be administered intravenously can besimilar to levels used in oral administration. Intravenous infusion maybe more controlled and accurate than oral administration.

Methods of calculating the frequency by which the composition isadministered are well-known in the art and any suitable frequency ofadministration can be used within the context of the present invention(e.g., one 6 g dose per day or two 3 g doses per day) and over anysuitable time period (e.g., a single dose can be administered over afive minute time period or over a one hour time period, or,alternatively, multiple doses can be administered over an eight weektime period). The combination of HMB and Vitamin D can be administeredover an extended period of time, such as months or years.

It will be understood by one of ordinary skill in the art that HMB andVitamin D do not have to be administered in the same composition toperform the claimed methods. Stated another way, separate capsules,pills, mixtures, etc. of Vitamin D and of HMB may be administered to asubject to carry out the claimed methods.

Any suitable dose of HMB can be used within the context of the presentinvention. Methods of calculating proper doses are well known in theart. The dosage amount of HMB can be expressed in terms of correspondingmole amount of Ca-HMB. The dosage range within which HMB may beadministered orally or intravenously is within the range from 0.01 to0.2 grams HMB (Ca-HMB) per kilogram of body weight per 24 hours. Foradults, assuming body weights of from about 100 to 200 lbs., the dosageamount orally or intravenously of HMB (Ca-HMB basis) can range from 0.5to 30 grams per subject per 24 hours.

The amount of Vitamin D in the composition can be selecting an amount ofVitamin D within the range of greater than 500 IU, as the below examplesindicate that 500 IU is the lower threshold for an effective amount inindividuals with inadequate levels of Vitamin D in the bloodstream, yetnot too much Vitamin D as to be toxic. While the examples indicate athreshold of 500 IU, lower amounts such as 400 IU, may be appropriate,in some individuals, to raise blood Vitamin D levels to an appropriateamount. In another embodiment, the amount of Vitamin D in thecomposition can be selecting an amount of Vitamin D within the range ofgreater than 400 IU, yet not too much Vitamin D as to be toxic. Thetoxic level of vitamin D is a person-specific amount and depends on aperson's blood level of vitamin D. For example, administration of100,000 IU of vitamin D may be toxic for healthy individuals, but nottoxic for a person suffering from rickets. One of skill in the art willrecognize toxicity levels for an individual. Further, the compositionmay include Vitamin D in amounts sufficient to raise blood levels ofVitamin D to at least around 25 ng/ml.

In the preferred embodiment, the composition comprises HMB in the formof its calcium salt, and Vitamin D in the form of 25-0H Vit D₃.Preferably, a composition in accordance with the present inventioncomprises HMB in an amount from about 0.5 g to about 30 g and Vitamin Din an amount greater than 500 IU, but not in an amount high enough to betoxic. One range of Vitamin D in accordance with this invention isaround 1000 IU to around 4000 IU. For examples, 1001 IU, 1002 IU, 1003IU . . . 1995 IU, 1996 IU, 1997 IU, 1998 IU, 1999 IU, 2000 IU, 2001 IU,2002 IU, 2003 IU, 2004 IU, 2005 IU . . .3997 IU, 3998 IU, 3999 IU, andall numbers between around 1000 IU and 4000 IU and not otherwise stated.

In another example, a range of Vitamin D in accordance with thisinvention is around 400 IU to around 100,000 IU. The specific amount ofvitamin D that is appropriate to administer to a particular individualroutinely varies. A healthy individual likely requires supplementationwith vitamin D in an amount lower than an individual with certaindisease conditions. For example, it would be appropriate in somecircumstances to administer vitamin D to an individual with rickets inan amount of 100,000 IU daily. One of skill in the art is able toreadily determine the amount of vitamin D that should be given to aparticular individual without causing toxicity.

The amount of vitamin D used in the present invention depends on theindividual's vitamin D status. In some individuals, around 400-5001U ofvitamin D is all that would be required to achieve a serum blood levelof around 25 ng/ml. In others, 2,000, 4,000 or even 100,000 IU ofvitamin D may be required. For example, 400 IU, 401IU, 405 IU, 450 IU,500 IU, 550 IU, 1000 IU, 1001 IU, 2000 IU, 5000 IU, 10,000 IU, 20,000IU, 50,000 IU, 75,000 IU and 100,000 IU and all numbers around andbetween 400 IU and 100,000 IU that have not been otherwise stated areincluded in this invention.

The Food and Nutrition Board at the Institute of Medicine of TheNational Academies has developed intake reference values for Vitamin Dand other nutrients. These values include the Recommended DietaryAllowance (“RDA”), which is defined as the average daily level of intakesufficient to meet the nutrient requirements of nearly all (97%-98%)healthy people; and Adequate Intake (“AI”), which is established whenevidence is insufficient to develop an RDA and is set at a level assumedto ensure nutritional adequacy. The RDA for Vitamin D is currently setat 600 IU, or 15 mcg, for males and females ages 1-70. For people overthe age of 70, the RDA is set at 800 IU of vitamin D (20 mcg). Forbabies from 0-12 months, an AI has been established of 400 IU (10 mcg).

Daily Values (DVs) are established by the Food and Drug Administration(FDA) and are used on food and dietary supplement labels. DVs suggesthow much of a nutrient serving of the food or supplement provides in thecontext of a total daily diet. DVs are presented on food and supplementlabels as a percentage. The Daily Value for Vitamin D, based on acaloric intake of 2,000 calories, for adults and children age 4 years ormore, is 400 IU. The Daily Value for Vitamin D is also 400 IU forinfants, children less than 4 years old, and pregnant and lactatingwomen.

These amounts are determined such that a large percentage of thepopulation taking these amounts will have sufficient Vitamin D levels.Heaney et al. have determined that a dose of 400 IU per day will elevateserum 25(OH)D₃ levels by 7.0 nmol/L (or 2.8 ng/mL) (99).

In one example of the present invention, the amount of vitamin D usedcan be expressed in terms of the Recommended Dietary Allowance (RDA),Adequate Intake (AI), and/or Daily Value (DV). For example, the presentinvention includes compositions of HMB and Vitamin D in an amount aroundat least as much as the Recommended Daily Allowance of RDA; compositionsof HMB and Vitamin D in an amount around at least as much as the DailyValue; and compositions of HMB and Vitamin D in an amount around atleast as much as the Adequate Intake.

The amount of vitamin D needed to reach appropriate blood serum levelsof vitamin D in accordance with the present invention may routinely varyfrom person to person, and determination of the optimum amount in eachinstance can be readily obtained by routine procedures.

In an additional embodiment, the composition in accordance with thepresent invention comprises HMB in an amount from about 0.5 g to about30 g and Vitamin D in an amount sufficient to increase circulating bloodlevels of 25OH-VitD3 or 25-OH VitD₂, depending on the form supplemented,to at least about 25 ng/ml.

In general, an amount of HMB and vitamin D in the levels sufficient toimprove overall muscle strength, function, and overall mass isadministered for an effective period of time.

The invention provides a method of administering a composition of HMBand Vitamin D to an animal such that the animal's muscle mass increases.The animal may or may not engage in exercise. Exercising in conjunctionwith the administration of HMB and Vitamin D results in an even greaterimprovement in strength and muscle function, but exercise is notnecessary to improve strength and muscle function. The amount of HMB andVitamin D in the composition administered that are effective forincreasing the animal's muscle mass can be determined in accordance withmethods well-known in the art. In one embodiment, the effective amountof HMB in the composition may be from about 0.5 g to about 30 g and theeffective amount of Vitamin D in the composition may be from greaterthan about 500 IU per 24 hour period. In another embodiment, theeffective amount of HMB is the same, and the effective amount of VitaminD is that which is sufficient to increase blood levels of Vitamin D toat least about 25 ng/ml.

The invention provides a method of administering a composition of HMBand Vitamin D to an animal such that the animal's strength increases.The animal may or may not engage in exercise. The amount of HMB andVitamin D in the composition administered that are effective forincreasing the animal's muscle mass can be determined in accordance withmethods well-known in the art. In one embodiment, the effective amountof HMB in the composition may be from about 0.5 g to about 30 g and theeffective amount of Vitamin D in the composition may be from greaterthan about 500 IU per 24 hour period. In another embodiment, theeffective amount of HMB is the same, and the effective amount of VitaminD is that which is sufficient to increase blood levels of Vitamin D toat least about 25 ng/ml.

The invention further comprises a method of administering a compositionof HMB and Vitamin D in an effective amount for improving musclefunction. The amount of HMB and Vitamin D in the compositionadministered that are effective for increasing the animal's muscle masscan be determined in accordance with methods well-known in the art. Inone embodiment, the effective amount of HMB in the composition may befrom about 0.5 g to about 30 g and the effective amount of Vitamin D inthe composition may be from greater than about 500 IU. In anotherembodiment, the effective amount of HMB is the same, and the effectiveamount of Vitamin D is that which is sufficient to increase blood levelsof Vitamin D to at least about 25 ng/ml.

EXAMPLES

The following examples further illustrate the invention but should notbe construed as in any way limiting its scope. For example, the amountsof HMB and Vitamin D administered and the duration of thesupplementation are not limited to what is described in the examples.

These examples demonstrate the surprising result that the combination ofvitamin D and HMB improves strength and muscle function. It waspreviously known that HMB supplementation increases muscle mass, but nocorresponding improvement in strength and muscle function was seen withHMB alone. The examples demonstrate that when serum levels of Vitamin Dreach appropriate levels, most typically through supplementation, musclestrength and function improve. The increases in strength, muscle mass,and improved muscle function described and observed in the examplesbelow demonstrate that HMB and Vitamin D are synergistic; when vitamin Dlevels reach an adequate amount, administration of HMB works better,more effectively or more efficiently than HMB when administered withoutadequate vitamin D levels. A composition containing HMB and Vitamin D insufficient amounts will be more efficient and more effective than acomposition containing HMB that does not also include adequate amountsof Vitamin D. The studies below examine the effects of vitamin D levelson the efficacy of HMB as related to muscle function, strength andmuscle mass, but the improved efficacy of HMB as described in thisinvention includes all known uses of HMB, including but not limited tothe use of HMB for disease associated wasting, aging, cachexia, andnitrogen retention. Further, the efficacy of HMB as related to immunefunction and lowering cholesterol are also within the scope of thisagreement.

Example 1

It is known that administration of HMB and amino acids, specificallyarginine and lysine, administered to the non-exercising populationresults in significant increases in lean mass and improvement in proteinturnover. Due to the increases in lean mass and improvement in proteinturnover, it would be expected that administration of HMB and aminoacids would also improve strength and function. Administration of HMBand amino acids alone, however, does not result in improvement instrength, function, or both. Instead, a gradual loss of handgrip and legstrength is observed. The following example demonstrates that the amountof Vitamin D in the bloodstream affects whether administration of HMBshows improvement in muscle strength and/or function. The datademonstrate that administration of HMB, arginine, and lysine(HMB/ARG/LYS) and Vitamin D is superior to administration of eitherHMB/ARG/LYS alone or Vitamin D alone. These results show a synergisticeffect between HMB and Vitamin D for improving muscle strength andfunction.

Methods

The effects of HMB, arginine, and lysine (HMB/ARG/LYS) on strength andmuscle function in the elderly was studied in both elderly men (n=38)and women (n=39) with an average age of 76.0±1.6 years (80). Subjectswere randomly assigned to either treatment with HMB/ARG/LYS (n=40) or toan isonitrogenous control group (n=37) for a one year study. Supplementswere taken once per day in the morning with breakfast and allsupplements supplied 0.1 g ascorbic acid per day. The HMB/ARG/LYScontained 2 g CaHMB, 5 g arginine, and 1.5 g lysine. The controlcontained 5.6 g alanine, 0.9 g glutamic acid, 3.1 g glycine, and 2.2 gserine. The supplements were supplied as a ready to mix orange-flavoredpowder. Subjects weighing more than 68 kg were given supplementscontaining 1.5 times the dosage above to maintain close to 38 mg/kg bodyweight per day CaHMB intake as this has been shown to be an effectiveintake of HMB per day. Lean tissue mass was measured using twoindependent methods, bioelectrical-impedance analysis (BIA) and dualenergy x-ray absorptiometry (DXA). Strength was measured in the upperand lower extremities.

The retrospective analysis of serum 25OH-VitD3 in the same cohort wasperformed and the data were stratified based upon the Vitamin D statusof the subjects within each original treatment group. An “adequateVitamin D status” was defined as serum 25OH-VitD3 level of ≥30 ng/mL(82-84). Consequently four cohorts were identified: (1) control-subjectswith a 25OH-VitD3 level <30 ng/mL (n=25); (2) HMB/ARG/LYS subjects witha 25OH-VitD3 level <30 ng/mL (n=29); (3) control subjects with a25OH-VitD3 level >30 ng/mL (n=12); and (4) HMB/ARG/LYS subjects with a25OH-VitD3 level >30 ng/mL (n=11).

Statistics

The data, means and changes in means over the 12-month period, wereanalyzed using a mixed models procedure of the Statistical AnalysisSystem for Windows (Release 8.02, SAS Institute, Cary, N.C.). A repeatedmeasures analysis of variance (ANOVA) that included the initial time 0values for the variable measured as a covariate and main effects ofsite, gender, and treatment. Lean body mass was analyzed using linearand quadratic time by treatment contrasts.

Results and Analysis

Supplementation with HMB/ARG/LYS resulted in statistically significantincreases in muscle mass irrespective of the Vitamin D status (FIG. 1a). The 12-month increase in lean mass within the cohorts with25OH-VitD3 >30 ng/mL was 0.9 kg, and 0.7 kg in the cohorts with <30ng/mL (FIG. 1a ), which were not significantly different. However,regardless of Vitamin D status, there was a significant increase in leanmass over the year long supplementation (FIG. 1a inset, p=0.02). On theother hand there was significant divergence in muscle strength basedupon Vitamin D status (FIG. 1b ). Measurements of “Overall KneeStrength” showed that the HMB/ARG/LYS-supplemented group with 25OH-VitD3level >30 ng/mL had significant improvements in muscle strength. Whenthis group was compared with the other three cohorts, there was a 21%linear increase in strength (p<0.003).

Taken together, these results suggest a synergistic effect between theHMB-supplemented cocktail and Vitamin D. Although HMB/ARG/LYSsupplementation increased muscle mass (fat-free-mass) regardless ofVitamin D status, strength was only increased with HMB/ARG/LYSsupplementation when subjects had adequate Vitamin D status.

The combination of HMB/ARG/LYS and adequate Vitamin D status isnecessary for and superior to either HMB/ARG/LYS alone or adequateVitamin D alone based upon the controls with adequate Vitamin D inimproving the strength and functionality of muscle. These results show asynergistic effect between HMB and Vitamin D for improving musclestrength and function.

Prior studies examining the effect of HMB on increasing muscle mass werenot significantly improved in all study designs (published andunpublished). It is believed that Vitamin D status may not have beenadequate to maximize muscle mass gains in these earlier studies in theHMB-supplemented subjects. This may be especially true in populationswhere low vitamin D status due to age, geographical location, dietaryintake, or disease was in question. Consequently, supplementation with acombination of HMB and Vitamin D may not only increase muscle strengthand functionality but also restore or increase muscle mass compared tosupplementation of HMB alone.

Example 2

In this example, subjects were administered a combination of HMB andvitamin supplements.

Materials and Methods: Elderly women (n=30) and men (n=16) wererecruited into a double-blinded controlled study. The older adults wererecruited from two locations in South Dakota: Brookings and Sioux Falls.The subjects underwent an initial screening and were randomized totreatments. Testing consisted of an initial (0 weeks) and follow-uptesting at 4, 8 and 12 weeks over the course of the 12-week study.

Prior to the start of the experimental period, we randomly assignednutritional supplements to each subject using computer-generated randomnumbers in a double-blind fashion. Treatments were arranged in a 2×2factorial design with two levels of HMB (0 & 3.0 g/d) and two levels ofVitamin D (0 & 2,000 IU/d). We assigned subjects to one of the followingfour treatments:

-   -   (1) Control    -   (2) HMB (calcium salt), 3.0 g/day    -   (3) 2,000 IU Vitamin D/day    -   (4) HMB, 3.0 g/day +2,000 IU Vitamin D.

The treatments were supplied in capsules of equal size and color andcontained equal amounts of calcium and phosphorus. The subjects wereinstructed to take three capsules two times a day. Each subject wassupplied with a one-week supply of the supplement, allocated by subjectnumber and returned each week for an additional 1-week supply.

The exercise testing and training session was supervised by trainedresearch associates. Resting heart rate and blood pressure measurementswere obtained prior to strength measurements. Enrolled individualsparticipated in an exercise training program consisting of strengthtraining exercises with Theraband® stretch cords (resistance training)and jumping. The equipment consisted of items that can easily be used athome. Exercise sessions were 3 times a week for 12 weeks. Each exercisesession was about 45-60 minutes. Testing sessions were performed at 0,4, 8, and 12 weeks. Each testing session lasted ˜60 minutes.

The strength program incorporated the following exercises: bicep curls,triceps extensions, chair squats, calf raises, ankle dorsiflexion,shoulder front raises and lateral raises, latissimus dorsi pull-down,chest press, seated row, knee flexion and extension, and hip flexion.For each of the 12 exercises, the participants completed 3 sets ofisotonic movement, 2 sets of 20 repetitions, and a final set to failure.When the 3^(rd) set could be performed for 20 repetitions in good form,the resistance was increased by moving to the next color of theresistance band. Between each set, participants performed a set of hopsor small jumps. Initially, 5 hops/jumps were performed following eachset. The number of hops/jumps was increased by 5 every 3 weeks until 25hops/jumps were achieved. Subjects remained at 25 hops/jumps betweensets for the remainder of the study. The number of hops/jumps wasreduced or omitted if there are any complaints regarding joint pain. Theresistance bands had been shown to safely increase strength andfunctionality when used in an older adult population (87-90).

Body composition measurements were obtained at 0, 4, 8, and 12 weeks.Measurements of strengths of quadriceps (extension/flexion) and elbows(extension/flexion) were obtained using the BIODEX IsokineticDynamometer. Handgrip strength was measured using a handgripdynamometer. Peak torque for knee extension and flexion was measured at60, 90 and 120°/sec. Peak torque for elbow extension and flexion wasmeasured at 60 and 120°/sec.

Functionality tests included: “Get-up-and-Go” performance (speed andgate), and “Get-up” performance. The “Get-up-and-Go” test consisted oftimed measurements of the subject's starting from a seated position,standing, walking forward 3 meters, turning around, walking back to thechair, and sitting down. The “Get-up” test consisted of the subjectstanding upright from a seated position as many times as possible within30 seconds. The complete descriptions and standardization of the testsare outlined in the Physical Dimensions of Aging by Waneen W. Spirduso(Human Kinetics, 1995).

A muscular performance index was calculated by summing the percentagechange from baseline for Get-up-and-Go, Get-up, handgrip, peak torquefor knee extension and flexion (60, 90 and 120°/sec) and peak torque forelbow extension and flexion (60 °/sec).

At 0, 4, 8, and 12 weeks blood samples were taken from a superficial armvein into Vacutainers™ (Becton Dickinson, Vacutainer Systems,Rutherford, N.J.) after an overnight fast. Serum was collected and usedto measure 25OH-VitD3 using a fully automated antibody-basedchemiluminescence assay (DiaSorin Inc., Stillwater, Minn.).

Least Squares Mean±SEM were calculated strength and functionalitychanges in each variable over the 12-week period. The mixed modelsprocedure of the Statistical Analysis System for Windows (Release 9.1,SAS Institute, Cary, N.C.) was used to analyze the data. Analysis ofCovariance was used with main effects of HMB, Vitamin D and theHMB*Vitamin D interaction. Data were adjusted using the initial serum25OH-VitD3 concentration as the covariate. The hypothesize was that thecombination of HMB and Vitamin D supplementation will result in agreater improvements in strength and functionality as compared tocontrol, HMB, or Vitamin D-treated subjects. This synergistic effect wastested with pre-planned one-tail t-test in a post-hoc analysis.Statistical significance was determined for p<0.05 and a trend wasdetermined for 0.05<p<0.10.

Results and Discussion: A total of 43 subjects completed the 12-week andone subject completed the 8-weeks of the study and was included in theanalysis. Two subjects dropped from the study prior to the 4-weekfollow-up visit and were not used in the analysis. The subjects thatdropped from the study did so because of the demanding commitment of a12-week training study and not due to any adverse events. The subjectcharacteristics of the 44 subjects used in the analysis are presented inTable 1.

TABLE 1 Subject Characteristics Item Control HMB VitD ₂₀₀₀ HMB +VitD₂₀₀₀ n 11 11 11 11 Age 73.4 ± 2.6 73.5 ± 3.0 72.1 ± 2.8 68.5 ± 2.5(62-87) (60-89) (61-96) (60-84) M/F 3/8 4/7 4/7 4/7 *25OH-VitD₃ 29.2 ±3.0 25.4 ± 3.0 24.0 ± 2.0 27.4 ± 3.0 (12.4-46.5) (12.2-39.7) (11.7-34.3)(15.1-42.9) #Leg Strength, 73.9 ± 9.6 64.1 ± 7.4  74.8 ± 10.0  83.1 ±10.6 Extension  (33-148)   (23-98.3) (24.7-133)   (40.5-146.1) Flexion51.4 ± 6.2 43.6 ± 5.3 55.9 ± 6.1 51.8 ± 7.7 (23-95) (14-61) (13.8-83)  (18-99) Data are expressed as Mean ± SE (min-max) *Baseline value#Baseline Peak Torque @ 60°/secThe average 25OH-VitD3 concentration at wk 0 was 26.5 ng/ml. Subjectstreated with 2000 IU of Vitamin D per day increased their serum25OH-VitD3 level by 10.7 ng/mL (FIG. 2a ). However, subjects eithersupplemented with a control or HMB alone decreased their serum25OH-VitD3 level by 2.0 ng/mL. A clean catch urine sample was used totest for supplement compliance. Subjects supplemented with HMB had a 50fold increase in urinary HMB concentration whereas the subjects notconsuming a HMB supplement had no increase urinary HMB concentration.

The effects of the combination of HMB and Vitamin D on knee extensionstrength (peak torque, 60°/sec is presented in FIG. 2b . Supplementationwith HMB+Vitamin D (7.28±5.03 nm) in older adults results in astatistically greater eight week increase in knee extension strength ascompared to control-treated subjects, P <0.05(−6.28±5.02 nm). Neitherthe HMB alone nor the Vitamin D-treated subjects increased kneeextension strength when compared to the control-treated subjects. Thesedata support our hypothesis that the combination of HMB+Vitamin D wouldbe synergistic on muscular strength.

Although not significant the performance index tends supports theseobservations (see FIG. 2c ). The performance index not only includes legstrength measurements but also functionality, hand grip strength, andelbow strength. These strength and function measurements are summed intoone overall performance index. The combination of HMB+Vitamin D resultedin a 146% increase in the performance index over the control. WhereasHMB and Vitamin D supplementation resulted in only a 35 and 58%increase, respectively, when compared to older adults supplemented withthe control treatment.

Implications: These data from a prospective study in older adultssupport the hypothesis that the combination of HMB and Vitamin Dsupplementation is synergistic, and resulted in significant improvementsin knee muscle strength and overall performance index. These datasupport the observation from our retrospective study in an elderlypopulation supplemented with HMB, Arginine, and Lysine. Muscularstrength increases only occur with HMB, Arginine, and Lysine in olderadults with adequate Vitamin D. In conclusion, the combination HMB andVitamin D is superior to either HMB or Vitamin D alone and is thereforesynergistic. These findings are important as the Vitamin D status inapproximately 66% of the elderly population is low and leads to adecrease in muscular strength and function. The loss of strength andfunction leads to an increase in falls and fractures, poor quality oflife and will ultimately impact health care costs.

Example 3

In this example, cell culture studies were performed to analyze theeffects of HMB and vitamin D on protein, DNA and protein turnover inmuscle cell cultures.

Recent in vitro studies have shown that HMB decreases protein breakdown.In one study HMB attenuated proteolysis-inducing factor (PIF) activationand increased gene expression of the ubiquitin-proteasome pathway inmurine myotubes, thereby reducing protein degradation (91). Otherstudies also suggest HMB may influence protein synthesis through themTOR pathway (92). Early studies have also suggested that vitamin Daffects protein metabolism (93). Vitamin D also binds a VDR found in thenucleus of muscle cells and regulates gene expression (94). Wehypothesize that HMB and vitamin D both have effects on muscle cells andthat if they act through independent mechanisms there could be asynergistic effect on either protein, DNA or protein turnover in thecells.

Methods

Mouse C2C12 skeletal muscle cell myoblasts were grown in culture understandard conditions and fused into myotubes following the methods ofMenconi et al. (95). Four separate studies were conducted duringdifferent weeks. The first 3 studies consisted of 4 replicate dishes pertreatment and the fourth study consisted of 6 replicate dishes pertreatment. The treatments were applied to the cells during the 24 hmeasurement period. Treatments applied were control, 25OH-VitD3 (200ng/mL), HMB (200 μM HMB as Ca(HMB)₂, and the combination of HMB and25OH-VitD3. Dexamethasone (100 nM) was added to the culture mediumduring the treatment/measurement period to stimulate proteindegradation. Calcium in the form of calcium chloride was added to thecontrol and 25OH-VitD3 treatments so that all the treatments werebalanced for calcium. For measurement of protein degradation during thetreatment period, leucine free medium was obtained to which[5,5,5]²H₃-leucine was added so that the isotopes of leucine (naturalleucine and [1-¹³C]-leucine) could be measured as they were releasedinto the medium through the protein degradation process. The measurementmedium was sampled at 2 and 24 h and samples stored at −80 ° C. untilanalyzed. The samples were analyzed for leucine using a gaschromatography (model 6890, Hewlett Packard, Palo, Calif.) massspectrometry (model 5973, Hewlett Packard, Palo, Calif.) and the gaschromatography column used for the analysis was a Zebron ZB-5.(Phenomenex, Torrence, Calif.). Natural leucine, [1-¹³C]-leucine, and[5,5,5²H₃]-leucine were monitored at 302, 303, and 305 AMU,respectively. Corrections for sampling at 2 h and for leucineutilization from the measurement media were made. The amount of theleucine isotopes released was also corrected for utilization(transamination and/or protein synthesis) (96). Concentrations ofproteins were analyzed following the microplate assay instructions usinga Pierce BCA protein assay kit (Thermo Scientific, Rockford, Ill.). DNAwas measured flourometrically using the Quant-iT™ dsDNA assay kit fromInvitrogen (Carlsbad, Calif.).

Statistics

Each cell culture dish was considered an experimental unit for analysis.Data presented are least square means and data were analyzed usingGeneral Linear Models in the Statistical Analysis System for Windows(Release 8.02, SAS Institute, Cary, N.C.). Main effects of experiment,HMB, vitamin D, and the interaction of HMB and vitamin D were includedin the model. Post-hoc least square means analysis was performed forindividual treatment means.

Results and Analysis

After the 24 h treatment period there were no treatment main effects ontotal protein content in the culture dishes. There were no significanttreatment main effects for DNA content. Protein:DNA ratio with thecombination of HMB and 25OH-VitD3 (FIG. 3) was significantly greaterthan either the control group or either treatment group alone and theinteraction was significant (p<0.003). This significant interactionresponse for the main effects would indicate a synergistic response ofHMB and 25OH-VitD3 on protein:DNA ratio. Measures of protein degradationand protein synthesis showed no significant main effects of treatment onprotein degradation measured either by natural or [1-¹³C]-leucinerelease into the medium from the cells. There was, however, a strongtrend for the interaction between HMB and 2=50H VitD₃ on proteindegradation measured by [1-¹³C]-leucine (p<0.08, FIG. 3). Post-hoc leastsquare means analysis of the effects of HMB alone showed a significantdecrease in protein degradation of 7.6 and 6.3% as measured by naturalleucine and [1-¹³C]-leucine, respectively (p<0.06). HMB and 25OH-VitD3combination treatment also decreased protein degradation, but the lackof neither a significant interaction nor a 25OH-VitD₃ main treatmenteffect would indicate the effect was due primarily to HMB. Proteinsynthesis and leucine utilization as measured by disappearance of[5,5,5-²H₃]-leucine from the media showed no differences amongtreatments. The current series of studies show that HMB and 25OH-VitD3combination increases protein:DNA ratio and that HMB decreases proteindegradation. These both are indications that the cell is producing moreproteins, possibly of a functional nature. The data also show that theHMB decreases dexamethasone stimulated protein degradation in bothslower and faster turnover protein pools natural and [1-¹³C]-leucinelabels, respectively. Vitamin D tended to decrease dexamethasonestimulated protein degradation in only the faster turnover protein pool.These could indicate that vitamin D affects synthesis of functionalproteins as vitamin D has been shown in other cell types to increasecell adhesion proteins (97, 98). In conclusion, the HMB +vitamin Dcombination was synergistic in increasing protein:DNA ratio in the cellsand supports our hypothesis.

Example 4

The amount of Vitamin D administered with HMB must be in an effectiveamount to raise the blood level of Vitamin D. In this example, it isdemonstrated that 500 IU of Vitamin D does not sufficiently raise theblood level of Vitamin D; this finding however, is based on the subjectsin this study. As stated hereinabove, the amount of Vitamin D necessaryto raise blood serum levels of Vitamin D to an adequate amount dependson the individual's Vitamin D status; in some instances, as little as400 IU of Vitamin D is an appropriate amount to raise blood levels toaround at least 25 ng/ml.

Subjects with adequate Vitamin D (plasma 25OH-VitD3 levels >30 ng/ml)manifested significant improvements in muscle function, while those withbiochemical evidence of Vitamin D deficiency (25OH-VitD₃ levels <30ng/ml) failed to show improvements in muscle strength and functionality.While muscular weakness associated with Vitamin D may not be surprisingat classical Vitamin D deficiency levels (blood 25OH-VitD3 of <15ng/mL), Bischoff-Ferrari et al continued to see improvement in lowerextremity function up to and beyond 40 ng 25OH-VitD3/mL which are levelswell above what previously might have been thought necessary for maximalbenefit (11). Therefore, supplementation Vitamin D should be adequate toraise 25OH-VitD3. FIG. 4a demonstrates the blood 25OH-VitD3 responsewhen a control, or supplement containing either 500 or 2000 IU ofVitamin D were given daily for 12 weeks in older adult over 65 years ofage. Subjects supplemented with either the control or 500 IU of VitaminD for 12 weeks did not increase blood 25OH-VitD3. Whereas subjectssupplemented with 2000 IU of Vitamin D increased blood levels by over 10ng/mL in 12 weeks. This supplementation established a blood level of25OH-VitD3 level >30 ng/ml. FIG. 4b shows the urine levels of HMB inthese subjects. In conclusion, 2000 IU of Vitamin D is sufficient toraise blood levels 25OH-VitD3, where 500 IU is inadequate. When VitaminD levels are adequately raised, the use of HMB results in an increase instrength and muscle function.

The foregoing description and drawings comprise illustrative embodimentsof the present inventions. The foregoing embodiments and the methodsdescribed herein may vary based on the ability, experience, andpreference of those skilled in the art. Merely listing the steps of themethod in a certain order does not constitute any limitation on theorder of the steps of the method. The foregoing description and drawingsmerely explain and illustrate the invention, and the invention is notlimited thereto, except insofar as the claims are so limited. Thoseskilled in the art who have the disclosure before them will be able tomake modifications and variations therein without departing from thescope of the invention.

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The invention claimed is:
 1. A method for increasing strength of ananimal in need thereof, the method comprising the steps of administeringto said animal a composition comprising amino acids and a synergisticcombination of from about 0.5 g to about 30 g ofβ-hydroxy-β-methylbutyrate (HMB) and Vitamin D in an amount sufficientto raise blood levels of Vitamin D to at least 25 ng/ml, wherein uponsaid administration of said synergistic combination of HMB and Vitamin Dto the animal, said strength is increased.
 2. The method of claim 1,wherein said HMB is selected from the group consisting of its free acidform, its salt, its ester and its lactone.
 3. The method of claim 1,wherein said HMB is a calcium salt.
 4. The method of claim 1, whereinsaid Vitamin D is Vitamin D₃.
 5. The method of claim 1, wherein saidVitamin D is Vitamin D₂.
 6. The method of claim 1, wherein saidcomposition is administered to said animal in an edible form.
 7. Themethod of claim 1, wherein said composition is administered to saidanimal intravenously.
 8. The method of claim 1, wherein the Vitamin D isgreater than 500 IU and less than or equal to 4,000 IU.
 9. The method ofclaim 1, wherein the amino acids are selected from the group consistingof arginine and lysine.
 10. A method for increasing strength of ananimal in need thereof, the method comprising the steps of administeringto said animal a composition comprising amino acids and a synergisticcombination of from about 0.5 g to about 30 g ofβ-hydroxy-β-methylbutyrate (HMB) and of from greater than 500 IU toabout 4,000 IU of Vitamin D, wherein upon said administration of saidsynergistic combination of HMB and Vitamin D to the animal, saidstrength is increased.
 11. The method of claim 10, wherein said HMB isselected from the group consisting of its free acid form, its salt, itsester and its lactone.
 12. The method of claim 10, wherein said HMB is acalcium salt.
 13. The method of claim 10, wherein said Vitamin D isVitamin D3.
 14. The method of claim 10, wherein the amino acids areselected from the group consisting of arginine and lysine.
 15. Themethod of claim 10, wherein said composition is administered to saidanimal in an edible form.
 16. A composition comprising amino acids and asynergistic combination of from about 0.5 g to about 30 g ofβ-hydroxy-β-methylbutyrate (HMB) and from greater than 500 IU to about4,000 IU of Vitamin D, wherein administration of the composition to ananimal in need thereof increases the strength of the animal.
 17. Thecomposition of claim 16, wherein said HMB is a calcium salt.
 18. Thecomposition of claim 16, wherein said Vitamin D is Vitamin D₃.
 19. Thecomposition of claim 16, wherein the amino acids are arginine andlysine.
 20. The composition of claim 16, wherein the composition is afoodstuff.